Recent work in non-transplant viral infection and autoimmune model systems has led to the understanding that variations in antigen (Ag) density during the priming phase of the immune response can have significant effects on the character and ultimate outcome of a T cell response. For example, evidence from the LCMV system demonstrates that the degree of viral load critically impacts the function and fate of the responding T cells, with increased viral loads being associated with reduced cytokine expression and eventual functional exhaustion. Increased exposure to Ag causes Ag-specific T cells to progressively lose function in a discrete hierarchical fashion. As a result, Ag-specific T cells are rendered unable to control the virus. In addition, studies in mouse models of autoimmune diabetes have revealed that increased Ag expression resulted in faster and more complete tolerance induction in the CD8+ T cell compartment. Conversely, relatively little is known about the instructional developmental T cell programs that are executed during allograft rejection and whether they are influenced by Ag density. While T cell exhaustion that occurs from chronic Ag exposure is unfavorable in a viral setting, this form of Ag-induced exhaustion may lead to favorable outcomes and increased donor-specific tolerance following transplantation. The issue of Ag density is very important in the study of transplantation, as the Ag density of allo-Ags may span a much broader range than that of nominal Ags. Therefore, we propose to characterize the effects of Ag density during the priming phase of the immune response on the function and fitness of the responding T cell population. Specifically, we will assess the effect of Ag density on the kinetics of skin graft rejection and programming of donor-specific CD4+ and CD8+ T cells, and determine the impact of variations in Ag density on the ability of CD28 and CD40L blockade to induce donor-specific tolerance and long-term skin graft acceptance. In addition, we propose to characterize the impact of Ag density on the kinetics of and requirement for PD-1 expression on Ag-specific T cells during tolerance induction. Lay Summary: Organ transplantation represents a curative therapy for many forms of end-stage organ disease. As surgical techniques for transplantation are improved, better strategies to prevent rejection of transplanted organs are needed in order to improve long-term graft survival, minimize the side-effects of anti- rejection medications, and reduce the need to re-engraftment. The research proposed in this application will provide much-needed insight into the mechanisms of transplant rejection, with the hopes of achieving these goals.